Method of producing tissue by placing a molding support within a body cavity

ABSTRACT

A method of producing a tissue includes placing a molding support within a body cavity for a time and under conditions sufficient for non-vascularized tissue comprising myofibroblasts to form on the molding support. In some embodiments, the tissue produced by this method is particularly useful as vascular tissue for the treatment or prophylaxis of diseased or damaged blood vessels such as in atherosclerosis.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. Ser. No.10/628,308, filed Jul. 29, 2003, which is a continuation-in-partapplication of U.S. Ser. No. 09/763,359, filed May 15, 2001, now U.S.Pat. No. 6,626,823, which is a 371 of PCT/AU1999/00670, filed Aug. 20,1999, the disclosures of which are hereby incorporated by reference.

FIELD OF THE INVENTION

The present invention relates generally to tissue implant material foruse in grafting procedures. More particularly, the present inventionprovides non-vascular tissue for use as vascular graft material. Thepresent invention further contemplates a method of vascular graftingusing non-vascular tissue.

BACKGROUND OF THE INVENTION

Bibliographic details of the publications referred to by author in thisspecification are also collected at the end of the description.

Tissue grafting represents a major advance in the medical treatment ofdiseased or damaged tissue. In some cases, tissue grafting representsthe sole avenue of medical treatment. However, the success of tissuegrafting depends on a range of factors including the availability ofsuitable donor tissue and the extent of immunological intolerance by therecipient.

An example of grafting is vascular grafting which is one approach indealing with atherosclerosis. Atherosclerosis is the principal cause ofheart disease, stroke and gangrene of the extremities. Atheroscleroticlesions are a result of an inflammatory response to a damaged arterywall and is associated with excessive lipid deposition (Schwartz et al,1993). The development of atherosclerosis (atherogenesis) is complex andinvolves several cell types such as macrophages, T-cells and smoothmuscle cells of the intima. Atherosclerosis is responsible for a highrate of mortality and an even higher rate of long term physicalimpairment of subjects affected by this disease.

A method of treating atherosclerosis is to insert bypass grafts aroundan artery blocked by plaques. The most common vascular graft material issaphenous vein or mammary artery from the patients. Such graft materialis referred to as an autograft. Vein and artery autografts are flexible,viable, non-thrombogenic and compatible. However, while the mammaryartery seldom develops atherosclerosis, it may not always be the propersize or length, and saphenous vein may have varicose degenerativealterations that can lead to aneurysm formation when transplanted to ahigh pressure arterial site. Furthermore, the non-thrombogenic surfaceof endothelial cells of saphenous veins is often damaged during graftpreparation.

Venous and arterial allografts have also been tried but have generallybeen abandoned clinically as they show a high incidence of rejection,deterioration and complications.

Similarly, the use of dialdehyde starch tanned bovine xenografts hasbeen generally abandoned due to a high incidence of aneurysm formationand poor resistance to infection.

For these reasons and because autologous grafts are not alwaysavailable, attempts have been made to produce synthetic vascularprostheses. The first synthetic vascular prosthesis was made of Vinyon-Nand was implanted into a patient in the late 1940's. The patient died 30minutes after the operation. Replacements have been made with nylon,then later with polytetrafluoroethylene (TEFLON) and polyethyleneterephthalate (DACRON). Nylon was found to lose most of its tensilestrength after a brief period of implantation leading to aneurysmaldilation and graft rupture. Although both polyethylene terephthalate(DACRON) and polytetrafluoroethylene (TEFLON) fabric grafts performreasonably satisfactorily in high flow, low resistance conditions suchas in the aorta, iliac and proximal femoral arteries, neither of thesetwo materials is satisfactory for small caliber arterialreconstructions. Such grafts are compounded by graft failures fromstenosis at the anastomic sites and excessive intimal hyperplasia. Thesecomplications are associated with graft thrombogenicity, poor healingand lack of compliance.

In the early 1970's, non textile vascular grafts prepared from expandedpolytetrafluoroethylene (ePTFE) were introduced. ePTFE is the mostchemically inert of all polymeric materials and is not degraded orchanged in the chemical environment of the body and is extremely easy tosuture. However, poor healing characteristics and lack of compliance aremajor causes for its lack of performance.

Indeed, the major problem with all synthetic vascular prostheses is thatthey are foreign bodies, so that blood coagulation can occur on theirluminal surfaces causing occlusion in prostheses. One innovationdesigned to improve the patency of the synthetic vascular graft is tocoat the lumen of the vascular graft with endothelial cells. While flowthrough the graft is improved and thrombogenesis reduced, graft failurecan still occur due to occlusion by overgrowth of endothelial cells. Inan attempt to control the growth, gene therapy has been used. Thisrefinement addresses the overgrowth, but retrovirally transduced cellson the graft are not able to withstand the shear stresses encountered byflow of blood and are sheared off. Also, the procedure for obtainingendothelial cells from the patient is invasive and the cells are hard topropagate in vitro.

Tissue-polymer prostheses are available which incorporate a combinationof tissue and synthetic material in the form of an integral composite.In one form, silicone mandrels covered with polyethylene terephthalate(DACRON) mesh are implanted beneath the cutaneous trunci muscles ofsheep where they become encapsulated with ovine collagen (Koch et al.,Aust NZ J Surg 67:637-639, 1997 1997). The tubes are then excised andtrimmed of excess fat and connective tissue is then fixed withglutaraldehyde. The silicone mandrel is then removed leaving thefibre-reinforced tube which, after sterilization, is stored in ethanol(Edwards and Roberts, Clin Mater 9:211-223, 1992). Although thisprosthetic device has been successfully used, it does suffer thedisadvantage of lacking elastin, an important component to preventaneurysmal and dilatory changes from stretching both the collagen andmesh components. Furthermore, the prosthetic device uses glutaraldehydeand this has the propensity to induce non-specific calcification of theimplanted device.

In summary, despite considerable experimental and clinical research,none of the biological and synthetic grafts produced thus far is anideal substitute for a blood vessel such as an artery, arterio-venousshunt or an access fistula. Limited availability, graft deteriorationand complications such as thrombosis, aneurysm formation and excessivesubintimal hyperplasia at the anastomotic sites are major problems.

There is a need, therefore, to develop tissue for use in vasculargrafting which exhibits the biocompatibility of a recipient's own tissuebut which is created artificially obviating the need to sacrificeexisting, i.e. indigenous, tissue from the recipient. In accordance withthe present invention, a means of producing living graft tissue for useas vascular tissue is identified but which is derived from non-vasculartissue.

SUMMARY OF THE INVENTION

Throughout this specification, unless the context requires otherwise,the word “comprise”, or variations such as “comprises” or “comprising”,will be understood to imply the inclusion of a stated element or integeror group of elements or integers but not the exclusion of any otherelement or integer or group of elements or integers.

The present invention contemplates the use of body cavities to generatetissue for vascular transplantation. The tissue may be used for exampleto replace a circulation vessel or part thereof or to bypass a blockedvessel.

The tissue is produced by introducing a foreign body such as a mouldingsupport, scaffold or other three-dimensional matrix to a body cavity,allowing time for granulation tissue to form on, around or in theforeign body, and then removing the foreign body together with thetissue from the body cavity. In one embodiment, the foreign bodyincludes or comprises a biodegradable scaffold. Alternatively, thetissue is removed from the foreign body.

Still another alternative, a combination of a biodegradable matrixassociated with or around a foreign body is employed. In this case, thebiodegradable matrix remains associated with the tissue when the tissueis removed from the other foreign body.

Accordingly, the foreign body such as a tube is generally separated fromthe support but a biodegradable matrix generally remains associated withthe tissue until it dissolves or breaks down. In an alternativeembodiment, the foreign body such as a tube is the biodegradable matrix.Tissue for vascular transplantation may or may not need to be everted.

The present invention also contemplates the use of body cavities togenerate vascular tissue on a moulding support such as a biodegradablematrix for vascular transplantation. The moulding support may be usedalone or in conjunction with another foreign body such as a tube whichis discarded prior to the tissue being used. Vascular tissues that maybe generated by application of the method of the present inventioninclude, for example, arteries and veins.

Still another aspect of the present invention provides a prostheticdevice which facilitates the provision of a foreign body such as amoulding support to a body cavity. Generally, although not exclusively,the prosthetic device comprises an elongated tubular member adapted tobe inserted into a body cavity. The elongated tubular member is furtheradapted to receive an inner elongated member such as a biodegradablescaffold or a mesh (e.g. polyglycolic acid (DEXON) coated tube of, forexample, polyethylene. The preferred form of the prosthetic device is amodified Tenckhoff Acute Peritoneal Dialysis Catheter although anysimilar device may be employed. The device enables growth of a tissuearound all or part of the inner elongated member in a controlled orsemi-controlled manner.

In a related aspect, the elongated tubular member has an outer sheath orsheath like member. In a preferred embodiment the outer sheath iscomprised of silicon. The outer sheath can be further adapted to haveperforations. In the preferred form, the perforations are arranged in aspiral spacing for the length of the sheath. In a most preferredembodiment, the perforations are about 2 mm in diameter.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an illustration of moulding support positioning in the ratperitoneum.

FIG. 2 is a diagrammatic representation of a cross section of agranulation (myofibroblast) tube showing (A) the tube as it appears onremoval from the body cavity with inner silastic tubing covered bylayers of myofibroblasts and collagen and coated with a single layer ofmesothelium; (B) the tube following removal of silastic tubing; and (C)the everted tube of living granulation tissue with mesothelium liningthe inside of the living tube, forming a structure resembling an artery.

FIG. 3 is a photographic representation showing A. Haemotoxylin andEosin (H&E) staining and B smooth muscle α-actin (dark) staining of therat myofibroblast tube after eversion.

FIG. 4 is a photographic representation of a transmissionelectronmicrograph of two mesothelial cells lining the lumen andmyofibroblasts in the wall of the tube.

FIG. 5 is a photographic representation of a low power transmissionelectronmicrograph of the full thickness of rat granulation tissue. Notemesothelial cell lining the lumen (left) and spindle-shapedmyofibroblasts throughout the wall.

FIG. 6 is a photographic representation of a transmission electronmicrograph of a macrophage within the rat myofibroblast tube.

FIG. 7 is a photographic representation showing A. Western blot forsmooth muscle α-actin in the presence and absence of γ-interferon. B.Staining for α-actin in RAW 264 and J774 macrophages treated withγ-interferon.

FIG. 8 is a photographic representation showing in situ hybridizationusing DIG-labelled Y-chromosome probe showing positive cells in the wallof the myofibroblast capsule formed in the peritoneal cavity of anX-irradiated female mouse transfused with male bone marrow cells.

FIG. 9 is a photographic representation showing A. Apparatus for graftstretching. B. Higher power showing grafts attached to hooks instretching apparatus.

FIG. 10 is a photographic representation showing: (A) insertion of anartificial artery into a rabbit carotid artery prior to releasing theclamps to permit blood flow; (B) an artificial artery functioning in arabbit carotid artery under pressure.

FIG. 11 is a graphical representation showing the number of α-actinstaining cells and their Vvmyo within myofibroblast grafts transplantedinto the rat abdominal aorta.

FIG. 12A is a graphical representation showing response of rat thoracicaorta (upper trace) and transplanted graft (lower trace) to 100 mM KCl(a) and 1×10⁻⁵ M acetylcholine (b).

FIG. 12B is a graphical representation showing response of rat thoracicaorta (upper trace) and transplanted graft (lower trace) tophenylephrine at 10⁻⁹ M(c) to 10⁻⁴ M(m)

c 1×10⁻⁹M phenylephrined 3×10⁻⁹M phenylephrinee 1×10⁻⁸M phenylephrinef 3×10⁻⁸M phenylephrineg 1×10⁻⁷M phenylephrineh 3×10⁻⁷M phenylephrinei 1×10⁻⁶M phenylephrinej 3×10⁻⁶M phenylephrinek 1×10⁻⁵M phenylephrinel 3×10⁻⁵M phenylephrinem 1×10⁻⁴M phenylephrine.

FIG. 13A is a photographic representation of a rabbit granulationcapsule around silastic tubing prior to transplantation.

FIG. 13B is a photographic representation of rabbit granulation tissueafter fixing and removal of the silastic tubing. The tissue has beentrimmed.

FIG. 14 is a photographic representation showing:

-   a. transverse section of a 10 mm tube of two week granulation tissue    four months after it had been grafted by end to end anastomoses into    the abdominal aorta of the same rat. Note the thickened    “adventitia”. Haematoxylin and eosin. X30.-   b. α-Smooth muscle actin staining (dark) of myofibroblasts in a 20    mm tube of two week granulation tissue formed in the rabbit    peritoneal cavity, four months after it had been grafted by end to    end anastomoses into the carotid artery of the same animal. Note    small blood vessels in “adventitia”. X100.-   c. Wall of 20 mm tube of two week granulation tissue formed in the    rabbit peritoneal cavity, four months after it had been grafted by    end to end anastomoses into the carotid artery of the same animal.    Stained with antibodies to smooth muscle myosin heavy chain (dark).    X150.-   d. Wall of 10 mm tube of two week granulation tissue formed in the    rat peritoneal cavity, four months after it had been grafted by end    to end anastomoses into the abdominal aorta of the same rat. Stained    with Weigert's elastic stain. Note elastic fibrils. X300.

FIG. 15 is a diagrammatic representation of a prosthetic device in theform of a modified Tenckhoff Acute Peritoneal Dialysis Catheter havingan outer elongated member with perforations to enable passage of cells,fluid and growth factors and an inner elongated member in the form of abiodegradable scaffold or mesh coated tube of polyethylene. The catheterfurther comprises a first flanged portion which is sutured to theperitoneal wall and a second flanged portion which is locatedsubcutaneously.

FIG. 16 A is a diagrammatic representative of a VASCAM device.

FIG. 16 B is a diagrammatic representation of the cross-section of theproximal end of a VASCAM device.

FIG. 16 C is a diagrammatic representation of the tilted view of theproximal end of a VASCAM device.

FIG. 16 D is a diagrammatic representation of a cross-sectional view ofthe distal portion of a VASCAM device.

FIG. 17 is a diagrammatic representation of the proximal end of a VASCAMdevice. All dimensions are given in millimeters (mm).

DETAILED DESCRIPTION OF THE INVENTION

The present invention is predicated in part on the surprisingobservation that granulation tissue produced in a cavity of a live bodyin response to foreign material is useful as grafting material. Thegranulation tissue comprises non-thrombogenic, mesothelial(endothelial-like) cells overlying several layers of myofibroblastswhich, in a preferred embodiment, is highly contractile, strong andresponds to agonists and antagonists in a manner similar to smoothmuscle in blood vessels. After grafting, elastic fibres are produced bythe myofibroblasts.

Accordingly, one aspect of the present invention provides isolatedtissue suitable for use in a vascular graft said tissue comprisinggranulation tissue produced on or around or in a moulding support.

Although this aspect of the present invention is directed to tissuesuitable for use in vascular grafting, the present invention extends tothe use of the non-vascular granulation tissue formed in a body cavityin any suitable graft. In a particularly preferred embodiment, thepresent invention provides living non-vascular tissue for grafting ofsubstitute blood vessels.

Accordingly, another aspect of the present invention provides anisolated substitute blood vessel or a portion thereof comprisinggranulation tissue covered by non-thrombogenic mesothelial cells whereinthe tissue forms on, or around or in a moulding support inserted into abody cavity of the intended recipient of the substituted blood vessel.

In one embodiment, the substitute vessel is a substitute for an artery.

In another embodiment, the substitute vessel is a substitute for anarterio-venous shunt or an access fistula.

Reference herein to “prior to use” means that prior to the tissue beingused in a vascular graft, such as a substitute blood vessel, it isremoved from the moulding support. This may occur immediately prior tografting or a period of time before grafting.

“Vascular” tissue refers to, for example, a replacement artery or veinformed with the assistance of a foreign body comprising or otherwiseassociated with a moulding support such as a biodegradable mesh,scaffold and/or matrix.

In particular, the moulding support may be referred to as a “foreignbody” which includes a form of scaffold or other three-dimensionalmatrix such as a chamber or pouch. The foreign body may also comprise orbe associated with a biodegradable matrix, mesh or other support.

The foreign body may also be a “spring-like” device.

In one case, vascular tissue is formed in association with abiodegradable support. This support may be the foreign body or thebiodegradable support may be a matrix surrounding all or part of theforeign body.

Accordingly, another aspect of the present invention provides isolatedtissue suitable for use in vascular tissue grafting said tissuecomprising granulation tissue formed on, around and/or in a foreign bodycomprising or in association with a biodegradable mesh or other support.

In a particularly preferred embodiment, the substitute artery isprepared in vivo by inserting a moulding support in the form of a tubeor biodegradable mesh or support in and/or into a cavity of a live bodyand maintaining the moulding support in vivo until such time asgranulation tissue forms on, around or in the molding. The granulationtissue takes the form of the shape of the molding. The moulding supportmay, in fact, become encapsulated. Preferably, therefore, where thetissue is for use as a substitute artery, the moulding is a hollow orsolid tube or wire mesh (including a spring-like tube) with a desiredlength and diameter. The moulding needs to provoke an inflammatoryresponse. In this regard, in a preferred embodiment, the mouldingsupport is recognised by the recipient as a foreign body. The mouldingsupport may or may not need to be sterile.

Although not wishing to limit the present invention to any one theory ormode of action, it is proposed that peritoneal or other body cavitymacrophages coat the moulding support together with other cells of theimmune system such as but not limited to cells involved in animmune-mediated inflammatory response. Cells proposed to be involvedinclude granulocytes, macrophages and stromal cells. The macrophageseventually take on a flattened appearance, and fibroblasts cells as wellas cells with an intermediate morphology appear. Eventually, acontinuous layer of mesothelial cells and cells resemblingmyofibroblasts forms. The cytoplasm of these cells shows the abundantrough endoplasmic reticulum seen in normal fibroblasts but also containsmassive but discrete bundles of microfilaments with dense bodies whichclosely resemble those of smooth muscle cells. This tissue is referredto herein as “granulation tissue”. The moulding support is then removedfrom the body cavity. In one embodiment, the moulding support isseparated from the tissue. In another embodiment to the moulding supportremains integrated with the tissue. This is particularly the case whenthe moulding support is a biodegradable mesh or spring-like tubularform. It may also be necessary in order to separate the cells from themoulding support (when required) to cut or sever parts or portions ofthe tissue. In the case of the preparation of a substitute artery, themoulding is in the form of a tube. However, a tubular mesh which isoptionally, spring-like is particularly preferred.

Accordingly, another aspect of the present invention provides anisolated substitute blood vessel or a portion thereof comprising atubular tissue section comprising living myofibroblasts withingranulation tissue wherein the tissue is formed on a tubular mould.

Yet another aspect of the present invention provides an isolated tissuesuitable for use in a vascular graft said tissue produced by the processof placing a moulding support within a body cavity for a time and underconditions sufficient for granulation tissue to form in, around or onsaid moulding support and then removing the moulding support from thecavity.

Still yet another aspect of the present invention contemplates a methodof producing substitute tissue for use in a vascular graft, said methodcomprising placing a moulding support within a body cavity for a timeand under conditions sufficient for granulation tissue comprisingmyofibroblasts to form in, around or on said moulding support and thenremoving said moulding support from the body cavity.

In a particularly preferred embodiment, the present invention isdirected to a method for producing a substitute blood vessel said methodcomprising inserting into a body cavity a moulding support in the formof a tube for a time and under conditions for granulation tissue withmyofibroblasts to form and then removing the tubular moulding from thebody cavity granulation tissue.

In one embodiment the tube is a solid tube. In another embodiment, thetube is in tubular mesh form or a spring-like. Where a particular shapeis required, these may be in planer ovoid, round, tubular, elongatedform amongst other forms.

Where the moulding support may also be known, as a scaffold or solid ormatrix support. It may be used to provide a desired shape or to providethe appropriate infrastructure such as a hollow core. Although thetissue may be removed from the solid support, this is not critical andeither a biodegradable solid matrix, such as a mesh or scaffold, may beused or the matrix may remain permanently in place. If the tissue isremoved, it may be advantageous to evert the tissue off the solidsupport, i.e. turn it inside out, such that the mesothelium lining thegranulation tissue is now lining the inside of the tissue, however, thepresent invention extends, in a preferred embodiment to non-evertedtissue. The foreign body may also comprise or be associated with abiodegradable matrix.

Any body cavity may be used including but not limited to the peritoneum,thoracic cavity, scrotum, brain, joint or pericardial cavity.Preferably, the cavity is lined with mesothelial cells. The peritonealcavity is the most convenient and least disruptive to the host and ispreferred in accordance with the present invention.

The moulding support may be surgically implanted into the body cavitywhere it is effectively placed without restraint in the cavity i.e. itis “free floating”.

Alternatively, the moulding support is fixed to a region within thecavity. This may make insertion and/or retrieval of the implant easier.For example, a moulding support may be provided by way of a catheter. Inthis regard, a moulding support such as a tubular moulding can beprovided to the peritoneal cavity, for example, via a prosthetic devicesuch as a peritoneal dialysis catheter. One example of a peritonealdialysis catheter is a Tenckhoff catheter. This provides a convenientmanner in which to gain access to the moulding in the peritoneum by aless invasive procedure than open surgical intervention. A catheter maybe employed as a source of tubular moulding per se, i.e. that piece ofthe catheter inserted into the cavity or the catheter may be used as aconduit for passing suitable moulding supports into and out of thecavity.

Accordingly, another aspect of the present invention provides aprosthetic device which facilitates the provision of a moulding supportto a body cavity, said prosthetic device comprising an elongated member,said member having a portion adapted to be inserted into a body cavityand a portion adapted to be external to the body cavity wherein theportion adapted to be inside the body cavity comprises a mouldingsupport or permits entry of a moulding support into said body cavitywherein granulation tissue forms in, on or around said moulding supportwhich granulation tissue is suitable for use as a vascular graft, suchas substitute blood vessel.

In accordance with this embodiment, the internal portion of theelongated member of the catheter may be the moulding support per se.Alternatively, the elongated member may be a hollow tube through which amoulding support may be passed from the portion of the catheter externalto the cavity to the portion of the catheter in the cavity. In the caseof the latter embodiment, the moulding support would preferably beextended past the terminal portion of the portion inside the cavity suchthat the moulding support or part thereof is exposed to the cavity.Conveniently, a line or wire or other means is attached to one part ofthe moulding support to facilitate retrieval of the moulding supportthrough the elongated member.

The portion of the member external to the body cavity may still belocated inside the body but outside the lining of the body cavity. Forexample, the external portion may be positioned subcutaneously.Alternatively, the external portion is outside the body.

Preferably, the body cavity is the peritoneal cavity.

Preferably, the elongated member is a filament or tubular mold.

Another aspect of the present invention provides a filament or tubularmould support capable of acting as a catheter for a body cavity whereinone portion of said filament or tubular mould support is present in thebody cavity and another portion of filament or tubular mould support isoutside the body cavity.

In one embodiment, the prosthetic device or filament or tube is packagedfor sale with instructions for use.

In one preferred embodiment, a Tenckhoff catheter or its functionalequivalent is used. This may have a single or double cuff ofpolyethylene terephthalate (DACRON) to prevent migration of bacteriaand, hence, peritonitis when used in the peritoneal cavity, and may beused with or without silicon discs to hold the omentum and bowel awayfrom the tubing. Conveniently catheters are inserted into the peritonealcavity over a guide wire through an incision, generally after firstinfusing with dextrose dialysis solution. The cuff is then sewn in placein the peritoneum and an adapter attached to the external portion of thecatheter. This allows peritoneal drainage or the continued addition offluid. The catheter is surgically removed after a suitable time (e.g.1-10 weeks such as 2-3 weeks) without damaging the granulation tissuecapsule.

In a most preferred embodiment, the prosthetic device is similar to thedevice shown in FIG. 15 and comprises an outer elongated member 1 whichis perforated such as with holes to facilitate passage of inter aliacells, fluid and growth factors into and out of the outer elongatedmember. An inner elongated member 3 comprising, for example, anelectrospun biodegradable nanofibrous scaffold or a polyglycolic acid(DEXON) mesh coat around a tube of polyethylene is removably positionedinside the outer elongated member. Sutures 4 in the peritoneal wall holdthe device in place and an external portion 5 ahead of a flanged region6 maintains the external portion on the skin surface.

In a preferred embodiment, the outer elongated member is comprised ofsilicon. In a most preferred aspect, the outer membrane is perforated orcontains holes. These holes or perforations may be in any arrangement orpattern. However, in a preferred aspect, the holes or perforations arearranged in a spiral arrangement for added strength of the sheath.

In a most preferred aspect, a hole or perforation is made in the outersheath and the sheath rotated at least about 5, 10, 15, 20, 25, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120,125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180° and a secondhole is made. This process is repeated until the desired number of holesor perforations are made. In a most preferred aspect, the outer sheathis rotated about 90° between the holes or perforations being made.

The holes or perforations may be any size, but most preferably they areat about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10,0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22,0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.30, 0.31, 0.32, 0.33, 0.34,0.35, 0.36, 0.37, 0.38, 0.39, 0.40, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46,0.47, 0.48, 0.49, 0.50, 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.557, 0.58,0.59, 0.60, 0.61, 0.62, 0.63, 0.64, 0.65, 0.67, 0.68, 0.69, 0.70, 0.71,0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.80, 0.81, 0.82, 0.83,0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.90, 0.91, 0.92, 0.93, 0.94, 0.95,0.96, 0.97, 0.98, 0.99, 1.00, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19 or 20 mm in diameter. In a preferred aspect, theholes or perforations are about 1 to 3 mm in diameter. In a mostpreferred embodiment the holes or perforations are about 1.9558 mm indiameter.

The hole or perforations may be any distance apart. In a preferredembodiment, they are about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9,1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 438, 1.9, 2.0, 2.1, 2.2, 2.3,2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7,3.8, 3.9, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5,10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 15.5,16.0, 16.5, 17.0, 17.5, 18.0, 18.5, 19.0, 19.5, or 20.0 mm apart. In amost preferred embodiment the holes or perforations are about 2 mmapart.

In one aspect, the perforations or holes are made along the entirelength of the outer elongated member or sheath. In a preferred aspect,the are no holes or perforations near the proximal or distal ends of theouter elongated member or sheath. In a more preferred aspect, the holesor perforations are at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8,0.9, 1.0, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,55, 56, 57, 58, 59, 60, 61 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72,73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,91, 92, 93, 94, 95, 96, 97, 98, 99 or 100 mm from the distal or proximalends.

The entire prosthetic device may be removed or just the inner elongatedportion. This enables a more controlled and less invasive approach togenerating vascular and non-vascular tissue with a reduced risk ofdeveloping scar tissue or occlusions.

Preferably, the prosthetic device is a modified Tenckhoff AcutePeritoneal Dialysis Catheter.

This approach may also be used for generating vascular tissue althoughit is a requirement that the tissue form around in, or on a mouldingsupport such as a biodegradable mesh or support.

Another aspect of the present invention provides a filament or tubularmould support capable of acting as a catheter for a body cavity whereinone portion of said filament or tubular mould support is present in thebody cavity and another portion of filament or tubular mould support isoutside the body cavity. Preferably, the filament or tubular portioncomprises an outer elongated member into which an inner elongated memberis inserted.

The present invention is particularly directed to the use of bodycavities to prepare the substitute vascular tissue. This is done,however, with the understanding that the present invention extends topreparing substitute tissue in vitro. For example, through tissueculture techniques including feeder layers, granulation tissue may beinduced to form on or around a moulding support. The use of in vitroculture techniques has an advantage in that culture conditions can bemanipulated and controlled such as by the addition of, for example,growth factors and cytokines. It also has the advantage of not requiringan invasive procedure in order to produce the artificial artery.Generally, an artificial vessel is made in vitro with no artificialsupport scaffold but with a scaffold of matrix it has created itself, aswith the mesothial-lined granulation tissue tube formed in a body cavityof a host.

The production of artificial vessels in vivo and in vitro both haveadvantages and both techniques are contemplated by the presentinvention. The moulding support is selected depending on the intendeduse of the tissue. For example, tubes, beads or discs may be used. Tubesare particularly useful for the preparation of substitute blood vessels.Mesh such as biodegradable mesh may also be used

The molding support may be any material including polymers such ascellulose, polyacrylamide, nylon, polytetrafluoroethylene (TEFLON),polyethylene terephthalate (DACRON), polystyrene, polyvinyl chloride,polypropylene, silastic tubing and polytetrafluoroethylene. As indicatedabove, it may also be biodegradable. The use of glass is alsocontemplated by the present invention but is not a preferred moldingsupport. Reference to “tubular molding” is not to be taken as limitingthe molding to a hollow tube. The present invention also contemplates amolding support in the form of a filament such as a solid fibre. Abiodegradable mesh or scaffold such as polyglycolic acid (DEXON) mesh isa particularly preferred material either as the foreign body or beingpart of, such as, surrounding the foreign body.

In a particularly preferred embodiment, the present inventioncontemplates a method for producing a substitute blood vessel saidmethod comprising inserting a moulding support in tubular form into theperitoneal cavity of a recipient for a time and under conditionssufficient for granulation tissue to form with myofibroblasts and theremoving the moulding from the peritoneal cavity.

The frame may be removed from the moulding support or maintained in,within or around the moulding support.

In one embodiment the moulding support is silastic tubing or itsequivalent. In another embodiment the tube is perforated such as in theform of a “syringe”. The length and diameter of the substitute bloodvessel is determined by the length and diameter of the tubing employedas the moulding support. Conveniently, the diameter of the tubing mayrange from about 0.1 mm to about 10 mm and more preferably from about0.5 mm to about 5 mm. The length of the tubing will depend on the amountof graft required and on the size of the body cavity. For example, alength of from about 0.1 mm to about 1000 mm, more particularly fromabout 1 mm to about 800 mm and even more particularly from about 3 mm toabout 500 mm may be employed. More preferably, the length is from about10 mm to about 250 mm such as 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55,56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107,108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119 120, 121,122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135,136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149,150 151, 152 153 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164,165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178,179, 180, 181, 182 183, 184, 185, 186, 187, 188, 189, 190, 191, 192,193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206,207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220,220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233,234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247,248, 249, or 250 mm. In addition, this procedure permits branched orlooped tubing being employed to generate branched or looped blood vesselgrafts.

The method of the present invention is particularly useful forgenerating substitute blood vessels since the substitute blood vesselsof the present invention exhibit a non-thrombogenic surface, havecompliance and elasticity, exhibit long-term tensile strength, arebiocompatible, are easy to handle and have suturability and areavailable in any size depending on the size and shape of the tubularmolding. The tubular moulding may also comprise spiral grooves. Thespiral orientation of smooth muscle cells in blood vessels facilitatescontrol of compliance.

The preferred recipient for the implantation of the moulding support isthe patient requiring the substitute blood vessel or transplant.However, it is within the scope of the present invention for substituteblood vessels or other transplantable tissue to be prepared in otherindividuals such as genetically related individuals or non-relatedindividuals. In the case of the latter, immune-suppressing therapy maybe required to effect the transplant.

The present invention is particularly directed to grafting in humansalthough the subject invention extends to other animals and birds suchas primates, laboratory test animals (e.g. mice, rats, rabbits, guineapigs), livestock animals (e.g. cows, sheep, pigs, horses, donkeys),companion animals (e.g. dogs, cats), captive wild animals, caged birds,game birds and poultry birds (e.g. chickens, geese, ducks, turkeys).

The present invention further contemplates the genetic manipulation ofthe substitute tissue. In one embodiment, once the tissue is removedfrom the body cavity, the mesothelial cells are transfected with a viralvector, naked DNA or other suitable genetic vehicle. Alternatively, orin addition, the myofibroblasts may be genetically manipulated.Generally, the aim of genetic manipulation is to introduce traits whichfacilitates function or operation of the graft. For example, genesencoding tissue plasminogen activator (tPA), urokinase plasminogenactivator (uPA) or streptokinase may be introduced. Alternatively, or inaddition, genes may be introduced such as which encode nitric oxidesynthase (NOS) which prevents unwanted clotting and spasm.

The present invention further contemplates a method of treatingatherosclerosis or other blood vessel disease said method comprisingby-passing or replacing the damaged blood vessel by grafting asubstitute blood vessel, said substitute tissue comprisingmyofibroblasts within granulation tissue.

Preferably, the substitute blood vessel is prepared by placing amoulding support comprising a tube in a cavity of a live body, such as aperitoneal cavity, for a time and under conditions sufficient forgranulation tissue comprising myofibroblasts covered by mesothelium toform, removing said moulding from the body cavity and separating themoulding away from the granulation tissue and then everting thegranulation tissue.

Yet another aspect of the present invention provides an isolated tissuesuitable for use in a vascular graft said tissue produced by the processof placing a moulding support within a body cavity for a time and underconditions sufficient for granulation tissue to form on or around or insaid moulding support.

Preferably, the tissue is suitable for use as a substitute blood vesselor a portion thereof, in which case the moulding support is in tubularform, including a tubular mesh form.

Preferably, the granulation tissue is covered by non-thrombogenicmesothelial cells. The granulation tissue generally comprises livingmyofibroblasts within granulation tissue. The living myofibroblastsproduce elastic fibres within a few weeks of transplantation to a highpressure arterial site. Elasticity is important to prevent aneurysmaland dilatory changes.

The present invention further provides an isolated substitute bloodvessel maintained in a frozen state for use by a mammal in which it isproduced said substitute blood vessel formed by placing a tubularmoulding within a body cavity of said mammal for a time and underconditions sufficient for granulation tissue comprising myofibroblaststo form and the removing said tubular moulding.

Preferably, the body cavity is the peritoneal cavity.

The formed tissue may be separated from the moulding support or it maybe integrated.

Still another aspect of the present invention contemplates the use of amoulding support in the manufacture of tissue suitable for use in avascular graft, said tissue comprising granulation tissue produced onsaid moulding support.

The present invention is now described with respect to the practice ofone particular preferred embodiment. The following description is in noway intended to limit the scope of the instant invention.

To prepare a substitute blood vessel, an approximately 20 to 100 mm longpiece of approximately 1 to 10 mm diameter silastic tubing comprisingspiral grooves is optionally coated with fibronectin which enhancesmacrophage adhesiveness. The tube is placed in the peritoneal cavity and10 to 15 ml of balanced salt solution and dextrose added together with agrowth factor or cytokine such as but not limited togranulocyte-macrophage colony-stimulating factor (GM-CSF) in order tostimulate macrophage recruitment and proliferation.

The peritoneal cavity is closed and in approximately 1 to 6 weeks andmore preferably 2 to 3 weeks later, the tube is removed from the cavity.

The present invention is further described by the following non-limiting

EXAMPLES Example 1 Creation of an Artificial Blood Vessel

The first step in creating an artificial blood vessel was to determinean appropriate implant material which would:

(i) initiate granulation tissue development;

(ii) be covered by mesothelium;

(iii) form a tube-like structure;

(iv) be of variable diameter and length;

(v) not attach to omentum/mesentery in the peritoneal cavity; and

(vi) allow its own easy removal from the granulation tissue.

The second step was to determine its optimal time for harvest.

a) Selection of Appropriate Material as an Arterial Template

Twenty male adult Wistar rats were anaesthetized with 2.5% v/v (O₂)halothane. A 20 mm incision was made in the shaved abdominal wall and avariety of objects—plastic silastic tubing (inner diameter range from0.5-5 mm), glass rod, expanded polytetrafluoroethylene (ePTFE) graft(inner diameter 5 mm) and polyethylene terephthalate (DACRON) graft(inner diameter 6 mm)—inserted inside the peritoneal cavity (see FIG. 1)then the incision closed by 8 interrupted sutures (10-0 polyglycolicacid (DEXON) silk). For comparison with previous studies, 10 ml boiled(rabbit) blood clot was inserted into some rats. Only one type of objectwas used per animal. Animals were divided into four groups (labelledGroups 1 to 4) corresponding to the length of time the foreign bodyremained inside the peritoneal cavity (Weeks 1 to 4, respectively).

When a boiled blood clot was placed inside the peritoneal cavity, themajority of the clot was reduced to a single ball suspended within theperitoneal cavity. The granulation tissue appeared as a circumferentialand organised layer of myofibroblasts on the outer surface of the clot.

Glass pipettes were found to be less suitable templates for artificialarteries since rats implanted with glass frequently had complicationsleading to death.

Polyethylene terephthalate (DACRON) graft was also found to be notpreferred as no organized pattern was found around the graft. Instead,there was a rather haphazard-like arrangement that penetrated the graftmaterial making it difficult to remove from the granulation tissuewithout tearing the tissue.

When ePTFE graft was implanted into the peritoneal cavity highlyorganized concentric layering of collagen and α-actin positive cellsoccurred. The main drawback with the ePTFE graft was the ease with whichit adhered to peritoneal fat bodies (omentum/mesentery). Subsequently, ahigh degree of vascularization was found on these grafts. As it wascritical for this study for the material to remain floating at alltimes, these grafts were rejected. As with the polyethyleneterephthalate (DACRON), difficulty also arose during separation of thegranulation tissue from the ePTFE graft, with large tears oftenoccurring.

The plastic silastic tubing was found to be the most effective materialas it had a greater than 35% rate of remaining afloat over theexperimental period. Close to the tubing there was a layer of connectivetissue covered by a layer of cell-rich granulation tissue. Mesotheliumformed an outer lining of the myofibroblast capsule. This mesotheliallayer is extremely important as it possesses fibrinolytic andanti-coagulant activity (Verhagen et al British Journal of Haematology95: 542-549, 1996). The silastic tubing was also the easiest to removefrom the granulation tissue, with little to no damage done duringharvesting. Most importantly, the tube-like structures of diameter 0.5to 5 mm could be easily everted such that the mesothelium now lined thelumen. This created a tubular structure that mimics the structure of anormal blood vessel, with an inner “endothelium”, “media” of smoothmuscle-like cells and outer “adventitia” of connective tissue (FIG. 2).The fact that tubes of such small diameter are producible is especiallyimportant since synthetic grafts are not suitable to replace vessels ofsmall caliber as their thrombogenic surface can lead to occlusion.

(b) Optimal Time for Harvest

Upon establishing the right material to be used for this study, theoptimal time for harvest was investigated. This was performed byhomogenization of the tube followed by Western-blot analysis todetermine the amount of smooth muscle α-actin protein in the granulationtissue at various times. The level of α-actin provides an indication ofthe number and degree of differentiation of myofibroblasts present inthe granulation tissue.

After one week, the smooth muscle α-actin protein level was close tothat in the abdominal aorta (97% of that in abdominal aorta), however,there was a variable thickness in granulation tissue. After two weeks,there was uniform thickening of the graft and the α-actin level was atits highest (106%). After three weeks, the level of α-actin haddecreased considerably (50%) compared to two weeks, post implant tissue.Histologically, there were few myofibroblasts present in the graft.After four weeks, there was the same amount of α-actin level as in thethird week post implant (46%), with relatively few myofibroblasts and athick capsule of connective tissue.

Therefore, the optimum time for the graft to be harvested from ratperitoneal cavity is 2 weeks post implantation. This ensures sufficientmyofibroblasts are present within the granulation tissue to make ahighly responsive wall against the high blood pressure encounteredfollowing transplantation of the everted tube of tissue into thearterial system.

Example 2 Myofibroblast Tubes can be Grown to Different Lengths and inDifferent Species

Having established that silastic tubing is a suitable mould to produce amyofibroblast tube, that tubes of different diameter (0.5 to 5 mm) couldbe produced, and that 2 weeks is the optimal period for theirdevelopment within the peritoneal cavity, the inventors next determinedwhether artificial arteries could be produced in a species other thanthe rat, and whether these vessels could be longer.

Four pieces of silastic tubing with outer diameter of 3 mm and length 10mm (rat) and 5 mm by 20 mm (rabbit) were placed inside each animal. Fivemale Wistar rats and five male New Zealand White Cross rabbits wereused. Two weeks after graft placement, animals were sacrificed. Thesilastic tubing was carefully removed and the tube of tissue gentlyeverted such that the mesothelial layer now lined the inside of thefreed myofibroblast tube. Segments of the four myofibroblast tubes(free-floating) from each animal were processed for transmissionelectron microscopy and light microscopy (Manderson and Campbell,Journal of Pathology 18: 77-87, 1986), while total protein was extractedfor Western Blot analysis (Hartig et al., Brain Res Protoc. 2(1):35-43,1997).

Rat aortae were used as control for the Western blot analysis and volumefraction of myofilaments (Vvmyo) and for the staining with labelledantibodies against cytoskeletal markers and contractile filaments.Densitometry was performed on these bands from both the 2 weekmyofibroblast tube and rat aorta to obtain a quantitative measure of theamount of protein present with the aid of the Mocha image analysissystem (Jandel Scientific). The bands were converted to values between0-255, depending on their intensity. The more dense and intense theband, the closer the value to 0 and the lighter and less dense the bandthe closer the value to 255. For each protein the results were expressedas myofibroblast tube protein relative to aorta % (see Table 1). Allstatistical analyses were performed using the statistical softwarepackage ‘SIGMA STAT’ (Jandel Scientific, Ca, USA). Comparison of datafrom Vvmyo studies was carried out with the paired Student's t-Test. Inall statistical analyses, a p value of less than 0.05 was consideredsignificant.

Haematoxylin and Eosin staining of both rat and rabbit tubes showed aconcentric layering of collagen bundles and spindle-shaped cells whichwere α-actin positive (FIG. 3). The inside lining of these tubes wascovered with a single layer of cells that stained positively for vonWillibrand Factor (Serotec). Transmission electron microscopy confirmedthe presence of mesothelial cells lining the inside of the myofibroblasttube (FIG. 4). In the outer region of the wall (which had been incontact with the silastic tubing), there was a layer of matrix with asingle layer of myofibroblasts. In the mid-portion of the tube, cells atdifferent stages were observed. Most cells were spindle shaped (FIG. 5).These cells had the characteristic of myofibroblasts with a foldednucleus indicating that they undergo contraction. Large amounts ofsynthetic organelles were present together with abundant focal/densebodies of contractile filaments. There were also cells that closelyresembled differentiated smooth muscle (FIGS. 4 and 5). The Vvmyo in thecells of the rat myofibroblast tube was 35.7% ″1.6% compared with 63.7%″5.7% (p<0.05) for smooth muscle cells in the aorta of the same animals.Macrophages, readily distinguished by their irregular shape and highvesicle content, were commonly seen around the edge of the tube (FIG.6). Western analysis also showed a relatively large number of ED1positive cells (marker for macrophage) in the myofibroblast tubecompared with the aorta.

Using cytoskeletal markers, Gabbiani and colleagues (see Sappino et al,Lab Invest 63:144-161, 1990; Desmouliere et al, Journal of Hepatology1995) described five different phenotypes of myofibroblast: V type(vimentin positive), VA type (vimentin & α-actin positive), VD type(vimentin & desmin positive), VAD type (vimentin & α-actin & desminpositive) and VADM type (vimentin, α-actin, desmin and myosin positive)cells. It was considered that V type cells resembled typical mesenchymalcells and VD cells corresponded to fibroblasts. The VADM type wasconsidered to have differentiated into smooth muscle while the VAD typewas associated with myofibroblasts. The myofibroblasts of the presentinvention expressed both vimentin and desmin and large amounts ofα-actin and β-actin. Thus, by these criteria, the cells in the implantare myofibroblasts, but tending towards smooth muscle. The myofibroblasttube also contained smooth muscle myosin heavy chain (a marker of smoothmuscle), but this was considerably less than in the aorta (Table 1).

The myofibroblast tube contained a similar amount of collagen Type I andIV as the aorta (Table 1). Fibres of collagen Type I tend to be quiteflexible and are strongly cross-banded, which makes it an idealconnective tissue. Collagen Type IV is present within the basal laminaand is important in forming a cell's anchorage to the main skeleton ofthe structure. Therefore, the collagen framework of the myofibroblasttube provides considerable stability and strength. The low level ofelastin indicates that the myofibroblast tube is more similar to amuscular artery than an elastic artery.

Thus, myofibroblast tubes of different diameter and length can beproduced, and in species other than the rat, which fulfill the 6required criteria for artificial arteries as outlined in Example 1above. The myofibroblast tubes have a marker indicative of macrophages(ED1), as well as markers consistent with myofibroblasts differentiatingtowards vascular smooth muscle. However, from these experiments it wasnot clear whether the myofibroblasts are actually derived fromperitoneal macrophages and whether they can be induced to differentiatefurther.

Example 3 Macrophages are the Source of Myofibroblasts/Smooth Muscle inthe Peritoneal Tubes of Tissue

a) In Vitro Studies

Cultures of macrophage cell lines (RAW 264 and J774) were grown inDulbecco's Modified Essential Medium (Gibco) and 10% v/v foetal calfserum at 371 C in 6% v/v CO2 humidified incubators. At sub-confluency,25 U/ml of γ-interferon (Holan Biotechnology) was added to themacrophages and incubated for 16 hours. This cytokine caused de novoexpression of SM α-actin proteins in both the RAW 264 (16%) and J774(13.5%) macrophages, as measured by Western blotting (Hartig et al, 1997supra) [FIG. 7].

This experiment indicates that γ-interferon induces pure populations ofmacrophages to express the smooth muscle contractile protein α-actin,which is not normally expressed by these cells. Thus, it may be possiblefor macrophages to be a source of cells containing contractile proteinunder certain inflammatory conditions.

(b) In Vivo Studies

Definitive proof that cells of haemopoietic origin (as are peritonealmacrophages) are the source of myofibroblasts in the peritoneal tubeswas shown by the following in vivo experiment: Female mice of the C57BL6strain expressing the Ly 5.1 antigen on the surface of cells ofhaemopoietic origin were X-irradiated (9Gy) to destroy all bone marrowthen immediately transfused with 106 bone marrow cells taken from thefemur of male C57BL6 mice of a congenic strain with haemopoietic cellsexpressing the Ly 5.2 antigen. Flow cytometry showed that there wasgreater than 80% repopulation of the donor (Ly 5.2) cells by 4 weeks.Into the peritoneal cavity of the host female mice was then placed aboiled blood clot or small piece of silastic tubing upon which a capsuleof granulation tissue formed within a few days. Staining with antibodiesto the Ly 5.2 antigen showed that the α-actin positive cells of thecapsule were derived from the donor and thus of haemopoietic origin.This was further substantiated by their positive in situ hybridizationwith a probe for Y chromosome, proving their source is male, and thus ofdonor bone marrow origin (FIG. 8).

Example 4 Myofibroblasts within Graft Walls can Differentiate FurtherTowards Smooth Muscle Cells

In the normal artery wall, smooth muscle cells are responsible formaintenance of vascular tone via contraction-relaxation and thecytoplasm of the cells is filled with myofilaments. However, followinginjury to the artery wall, the smooth muscle cells are responsible forrestoring vascular integrity through their proliferation and synthesisof extracellular matrix. To do this, the smooth muscle cell loses itscontractile ability (and contractile filaments) as the cytoplasm becomesfilled with organelles involved in synthesis such as rough endoplasmicreticulum and free ribosomes. That is, the cells temporarily lose theappearance of differentiated smooth muscle cells and become morefibroblastic in structure and function. This is called modulation ofphenotype (Campbell et al, Arteriosclerosis 9(5):633-643, 1989).

Fibroblasts are also able to modulate their phenotype in response toexternal cues. For example, during continuous tissue reorganizationprocesses such as in the ovarian follicles, pulmonary septa, intestinalmucosa and during wound healing, fibroblasts express contractileproteins such as α-actin and at these times the fibroblasts are said tohave differentiated into myofibroblasts.

The inventors investigated whether environmental factors affect thephenotypic (or differentiation) state of the macrophage-derivedmyofibroblasts which comprise the granulation tissue tubes formed in theperitoneal cavity in response to silastic tubing.

a) Neuronal Influences do not Affect the Differentiation ofMacrophage-Derived Myofibroblasts

Structures which are implanted into the anterior eye chamber becomerevascularized and reinnervated by the surrounding nerves of the host(Puchkov et al, Morfologia 110(5):15-19, 1996). Under these conditions,various developing tissues such as embryonic rat heart, fetal skin andadrenal medulla develop into their functional adult form.

To assess whether innervation induces further differentiation ofmacrophage-derived myofibroblasts, pieces of myofibroblast tube weretransplanted to the anterior eye chamber of the rat from which it washarvested. Two week old myofibroblast tubes were removed from theperitoneal cavity of 12 Wistar rats under anaesthesia as earlierdescribed. Segments of 1-2 mm in diameter and 5 mm in length were placedin phosphate buffered saline ready for the implantation. The eye of thesame animal in which the myofibroblast tube was grown was carefullyrinsed with distilled water and a drop of atropine applied to dilate thepupil. With the aid of a dissecting microscope, the cornea waspenetrated obliquely with a specially prepared razor blade. The graftwas inserted into the anterior eye chamber with sterile watchmakerforceps away from the pupil.

At termination (2 months post implantation) the rats wereperfusion-fixed with 4% v/v glutaraldehyde via the left ventricle. Theeyeball was dissected out and the implants were removed. Staining of themyofibroblast graft with sucrose-phosphate-glyoxylate (SPG) showed themajority of the innervation occurred along the periphery of the graft.There were signs of cell death (pyknotic nuclei) in and around thetransplant, and most of the bulk of the wall consisted of collagenousmatrix.

Contrary to expectation from previous studies with undifferentiatedtissue, there was no significant change in the volume fraction ofmyofilaments (Vvmyo) of the cells (33.3+2.7% compared to 35.9+2.3%,p>0.05, in the non-transplanted myofibroblast tube). This indicates thatinnervation does not induce further differentiation ofmacrophage-derived myofibroblasts towards smooth muscle.

(b) Mechanical Factors Such as Active Stretching Lead to FurtherDifferentiation of Macrophage-Derived Myofibroblasts

Two week old pre-implant myofibroblasts were obtained from theperitoneal cavity of male Wistar rats. Under sterile conditions theywere attached to the sterilized stretching apparatus (FIG. 9). At eitherend of the chamber are hooks (made from a syringe) to which the graft isattached. The hook closer to control box contains a retractile coil,which can be set to different frequency and amplitude, while theopposite end remains stationary. The culture medium was added to thechamber with the graft attached to the recoiling device. The wholechamber was placed inside the incubator (370 C) and the graft underwentcontinuous stretching for 3 hours, 24 hours and 72 hours. The graft wasstretched 50 times per minute to 105-110% of its resting length. At theend of the experiment the grafts were prepared for morphometric analysisof Vvmyo.

No signs of damage were observed while the graft underwent stretching.At the beginning of each stretching experiment, the myofibroblasts werelocated in random direction within the graft wall. Between 3 and 24hours stretching, the myofibroblasts aligned themselves along thedirection of the stretch and remained in this position following 72hours of stretching. Most of the cells appeared to be more spindle andnarrow compared to the control (pieces of the same grafts that were notstretched). Mural thickening could be seen at 72 hours of stretching,mostly composed of collagenous matrix.

No significant increase in the mean % Vvmyo was seen in 3 hours poststretching (44.5+6.1% compared with control in this experiment,42.3+3.4%). However, the Vvmyo was significantly higher (p<0.05)following 24 hours of stretching (58.3+4.2%). At 72 hourspost-stretching, the amount of myofilaments was reduced (48.8+3.9%) andreplaced with large amounts of rough endoplasmic reticulum consistentwith the observed increase in collagenous matrix.

From this study, the inventors conclude that cyclic and directionalstretching stimulate the myofibroblasts to realign in the direction ofthe longitudinal strain and differentiate further towards contractilecells (mature smooth muscle). As the stretching continues, themyofibroblasts need to secrete additional collagen into their matrix tostrengthen the graft and accommodate changes to their environment. Theythus modulate back towards a “synthetic” cell in a similar way thatvascular smooth muscle does in response to challenge to vessel wallintegrity (see earlier).

Example 5 The Myofibroblast Tube as an Autologous Vascular Graft(Artificial Artery)

Two animal models, the rabbit and the rat, were used to determine thepotential usefulness of the myofibroblast tube as a vascular graftmaterial. Animals suffer no adverse effects from induction andharvesting of multiple peritoneal cavity-derived capsules over severalmonths. The rabbit had a segment of its right carotid artery removed andreplaced with the myofibroblast tube, while the rat had its abdominalaorta cut but not removed and a myofibroblast tube inserted at the cutends. FIG. 10 shows different rabbit carotid arteries before and afterblood is permitted to flow through the substitute vessel.

a) Transplantation of Rat Myofibroblast Tube to the Rat Aorta

Thirty male Wistar rats (250 to 350 g), each containing a 2 week oldmyofibroblast tube in the peritoneal cavity, were divided into fivegroups (n=6). The rats were premedicated with atropine (0.25 mg/kg bodyweight administered intraperitoneally), and anaesthetized with 1% v/v(O2) halothane. Under sterile conditions, each animal was prepared foroperation by shaving the abdomen, and wiping off excess hair with cottongauze soaked in an antiseptic solution (Betadine). The myofibroblasttube was harvested from the peritoneal cavity of the same animal intowhose abdominal aorta it was to be grafted. Only free-floating capsuleswere used. The abdominal aorta was exposed by a midline abdominalincision and dissected free from the adjacent vena cava and surroundingtissue.

With the aid of an operating microscope, two vascular clamps were placedon the area above and below the transplant site. The abdominal aorta wasresected and elastic recoil of the arteries left a gap of 0.5-1 cmbetween the cut ends. The silastic tubing was removed from themyofibroblast tube and the graft everted, trimmed and aligned with thisgap ready for suturing. Two stay sutures (9-0 silk) were placed at eachanastomosis to orient the graft and the artery, and to facilitate theplacing of other sutures. The mid-anastomotic site was sutured with 10-0(22 Fm) Ethilon suture material and round and non-traumatic needles.Suturing at the distal anastomosis was done first, followed by theproximal anastomosis. A total of eight interrupted sutures were placedat each end: one each at the dorsal, ventral, medial and lateral aspectof the anastomosis. Four more sutures were then placed to fill theintervals between them. Interrupted sutures, Ethilon 9-0 (Ethicon, Inc.,Thornwood, N.J.) were used. If more sutures were made a tighteningeffect was seen at the anastomotic sites where there is potential for ananastomotic aneurysm. The use of stay sutures was important, since theyprevent accidental suturing of the front and back wall of the samevessel. The grafts were not preclotted, nor was heparin or spasmolyticsadministered.

When suturing was completed, the distal clamp was released to allow thegraft to fill with blood under low pressure, and then the proximal clampreleased to allow blood flow under full arterial pressure through thegraft. Light external pressure with Gelpro sealant was required at theanastomoses to control initial leakage. Haemostasis was achieved byabout 2-3 minutes after removal of the clamps, but the graft wascontinuously monitored for 10-14 minutes in case of secondary bleeding.Patency was determined by direct inspection. The intestines were thenplaced back and the wound irrigated with saline solution and closed withpolyglycolic acid (DEXON) 4-0 sutures. The rats had free access tostandard food and water. A graft was deemed successful at the time ofoperation if it had a fully dilated and pulsing appearance, and afemoral pulse was present. Unsuccessful grafts were usually limp andflaccid, with no detectable pulse.

Six rats from each group were sacrificed at the end of theirexperimental period (1, 1.5, 2, 3 & 4 months post-implant). At the timeof sacrifice, the rats were anaesthetised with Ketamin and Xylazine (1ml/kg body weight administered intraperitoneally). Patency was evaluatedin all transplants taken at 1, 1.5, 2, 3 and 4 months and tissue at 1.5months was taken for organ bath studies. Remaining tissue was fixed forhistological studies (see Example 6). Wall thickness was measured andcell density calculated with the Mocha (Jandel scientific) imageanalysis using the modified method of Kleinert et al Cell transplant5(4): 475-482 1996. This was done in all vessels post-transplantationand in trimmed segments of vessels pre-transplantation.

After 1 month and 1.5 months transplantation, grafts in all 6 rats ofGroup 1 had a pulse and were patent. At 2 months, 4 out of 6 grafts werepatent, and at 3 and 4 months there were 3 out of 6 patent grafts,giving an overall patency rate of 73% (Table 2). None of the grafts hadbeen preclotted nor was heparin or spasmolytics administered to theanimals at the time of transplantation as the anti-thrombotic benefitsof the mesothelial lining were being tested. Non-patent grafts had theirlumen blocked by incorporated thrombus and by α-smooth muscle actinstaining cells. Signs of recanalization were sometimes seen. The patentrat grafts possessed a normal, intact wall and a strong pulse.Mesothelium (or migrated endothelium) which stained for von Willebrandfactor comprised the inner lining. The average wall thickness of thegraft increased from 0.18″ 0.02 mm (pre-transplant) to 0.25″ 0.02 mm by1 month after which no further change was observed. No significant(p<0.1) increase in cell number was evident in the “media”, with theincrease in graft thickness due to the large amount of extracellularmatrix, mainly collagen, which developed on the outer surface of thewall. This “adventitia” contained vasa vasora as seen with antibodies toα-smooth muscle actin.

The cells within the wall of the grafts in the rat stained intensely forα-smooth muscle actin and smooth muscle myosin. By 3 months the Vvmyo ofthe cells in the rat transplant had increased to 58.7″ 1.4% which wasnot significantly different from smooth muscle cells in the rat aortanear to the transplant site (Table 4). Structures that resembled elasticlamellae and stained with both Hart's and Weigert's elastic stain beganto appear in the grafts by 1 month, at first only near the lumen thenthroughout the “media” (FIG. 14 d).

b) Transplantation of Rabbit Myofibroblast Tube to the Rabbit CarotidArtery

Twenty male New Zealand White cross rabbits (aged 3-4 months), eachcontaining 2 week myofibroblast tubes in their peritoneal cavity, werepre-anaesthetized with 1 ml of Saffan (i.v., Gloxovet, Victoria,Australia) injected into their marginal ear vein. Continuous anaesthesiawas achieved with 2.5% v/v (O2) halothane. The myofibroblast tube washarvested from the peritoneal cavity first. To expose the right carotidartery, a midline incision (approximately in line with the trachea) wasmade. The surrounding connective tissue was blunt dissected and thesubmandibular glands clamped and retracted to one side to clear theunderlying blood vessels. At all times the operated area was kept moistwith saline. The transplanting procedure was similar to that in the rat,however, a longer myofibroblast tube was utilised in the rabbit (20 mm)compared to the rat (10 mm). A 1 cm segment of the carotid artery wasresected and elastic recoil of the arteries left a gap of about 2 cmbetween the cut ends. The trimmed, everted graft myofibroblast tube(with silastic tubing discarded) was sown into place.

At termination, the animals were anaesthetised and the carotid arteryexposed and cleaned from the surrounding tissue. The patency of thetransplants was assessed as 70% (Table 2). The animals were perfusedthrough the left ventricle with 2.5% v/v glutaraldehyde. Grafts wereremoved and post-fixed in glutaraldehyde overnight before being placedinside the tissue processor for wax embedding. A similar histologicalappearance was seen in rabbit as described above in rat even thoughdifferent lengths and diameter of grafts were transplanted intodifferent arteries (abdominal aorta and carotid artery, respectively).The myofibroblasts in the wall of the artificial artery differentiatedfurther towards the phenotype expressed by vascular smooth muscle cells,with Vvmyo of about 60%. These cells expressed both α-smooth muscleactin (FIG. 14 b) and smooth muscle myosin heavy chain (FIG. 14 c). Aswith the rat transplants, elastin fibres developed one monthpost-transplantation.

These findings show that an artificial artery grown within the patient'sown peritoneal cavity (or any other cavity lined by mesothelium) may beused as an autologous arterial transplant into a high pressure site. Themyofibroblast graft possesses a living, anticoagulant surface(mesothelial lining) and a living, contractile wall(myofibroblasts/smooth muscle), with tensile strength provided bycollagen Type I.

This new type of graft material may open new perspectives in the fieldof arterial reconstructive surgery. A biosynthetic graft that is growninside a patient's own body ensures no tissue rejection and limitedgraft complication. It obviates the need for removing mammary artery orsaphenous vein (which are often varicose in the elderly) from thepatient, and allows the required diameter and length of graft (possiblybranched as well as straight) to be grown as a form of “designerartery”. Since several grafts can be grown at the same time, it allowsfor multiple bypass grafting with grafts of different diameter.

Example 6 The Myofibroblast Tube Forms a Living, Contractile Conduit

To determine whether the myofibroblast tube functionally behaves in thesame manner as the host artery into which it has been transplanted inresponse to contractile and relaxing agents, the following experimentswere carried out.

Myofibroblast tubes, grown in the peritoneal cavity of a rat for 2weeks, were harvested and transplanted into the abdominal aorta of thehost as described in Example 5 (a). One and a half months aftertransplantation, 3 rings from each graft and thoracic aorta from thesame animal were suspended on stainless steel wire hooks in a jacketedwater bath. After equilibrium conditions were achieved, 100 mM potassiumchloride (KCl) was added to the organ bath to determine whether thetransplanted graft had any contractile activity. The normal thoracicaorta had an increase in contraction of 16.0 mN while the transplantedgraft contracted to 3.8 mN (FIG. 12A). While the rings were contracted,acetylcholine at 10-5 M was added and both rings relaxed.

Both tissues were then exposed to the contractile agonist5-hydroxytryptamine (5-HT) from 10-9 to 3×10-5 M. While the thoracicaorta began contracting at 3×10-7 M and reached a peak of 19 mN at3×10-5 M, the transplanted graft had no contractile response.

Similar studies were carried out with phenylephrine at 10-9 to 10-4 M.The thoracic aorta began contracting at 3×10-9 to a maximum of 15.5 mNat 3×10-5 M, while the graft contracted at 10-6 M to reach a maximum of1.5 mN at 10-5 M (FIG. 12B).

When the myofibroblast tube was taken directly from the peritonealcavity after 2 weeks development and tested as above, there was noresponse to any agonist.

These studies demonstrate that the myofibroblast tube, several weeksafter transplantation into the host aorta, begins to acquire the sameresponse to contracting and relaxing agents as the bone fide artery.This has important clinical implications since it demonstrates that thegraft is capable of responding to the same circulating regulators ofvessel wall tone as the host artery.

Example 7 Improvements in Generation of the Myofibroblast Tube in theRabbit

The following improvements in the generation of the myofibroblast tubehave been made in the rabbit model:

Increase in length of myofibroblast tube

In 6 rabbits it was found that lengths of silastic tubing of 60-80 mmand 1.9 mm outer diameter could be used to form a myofibroblast capsule(FIG. 13A). These capsules were complete and of even thickness and wheneverted formed a tube of living tissue of the same diameter as thecommon carotid artery into which a 20 mm segment was sutured (FIG. 15B).

Another advantage of tubing of this longer length was that it formedfewer adhesions to the peritoneal fat or the bowel. This may be due tothe inability of the longer lengths (rather than 20 mm as previously inthe rabbit) to move deep into the peritoneal cavity amongst theintestines.

Addition of 1.5% w/v dextrose in balanced salt solution to theperitoneal cavity

A reduction in the rate of adhesion formation (ie fewer adhesions) wasachieved, in part, by the addition of 1.5% w/v dextrose in 10-15 mlbalanced salt solution added to the peritoneal cavity at the time oftube insertion.

Sterility

In the rat it had been found that thicker capsules of granulation tissuedeveloped if the tubing was unsterile. However, in the rabbit it wasshown that capsules of comparable thickness developed when the tubinghad been sterilized in 70% v/v ethanol and air-dried prior toimplantation in the peritoneal cavity.

GM-CSF in peritoneal fluid

The addition of 0.02 μg granulocyte-macrophage colony stimulating factor(GM-CSF) in 10 ml 1.5% w/v dextrose in phosphate buffered saline addedat the same time as tube implantation in the peritoneal cavity of therabbit resulted the development of a very uniform and thick capsule ofgranulation tissue. This was assumed to result either from an increasednumber of peritoneal macrophages recruited into the cavity or fromproliferation of existing peritoneal macrophages.

Spiral grooves in the silastic tubing

The spiral orientation of smooth muscle cells in blood vessels providesfor larger and better control of compliance compared with a structurewhere the cells and collagen fibres have formed a circumferential orlongitudinal array.

Under normal conditions the cells in the granulation tissue that formson silastic tubing in the peritoneal cavity is in a longitudinalorientation. In order to encourage the myofibroblasts to develop inspiral arrangement around the silastic tubing, rather than alongitudinal orientation, spiral grooves were etched into the tubingwith glass paper. This orientation of the myofibroblasts closelyresembled the organization of smooth muscle cells in blood vessels.

Coating of tubing

To determine whether coating the silastic tubing resulted in a thicker,even capsule the following substances were applied prior to implantationof the tubing in the peritoneal cavity:

a) Tubing was coated with plasma proteins by incubating sterile tubingin 100% foetal calf serum for 12 hours at 37EC, then thoroughly drainingthe tubing.

b) Tubing was coated with collagen Type I prepared from rat tail tendonsby dipping and drying 3 times.

c) Tubing was coated with fibronectin.

d) Tubing was coated with laminin.

Example 8 Improvement in Graft Patency

In Example 5, no anticoagulants or antiplatelet agents or spasmolyticswere given to the animals at the time of transplantation, before orlater, in order to test the thromboresistance of the mesothelial liningof the implant. Under these conditions, the patency rate was 73% in therat (30 rats at 1, 1.5, 2, 3, or 4 months post-transplantation) or 70%in the rabbit (20 rabbits at 1, 2, 3, or 4 months post transplantation(see Example 5).

In order to determine whether the addition of heparin improved thepatency rate in the rabbit, the following procedure was carried outimmediately prior to suturing 20 mm lengths of everted granulationtissue into the rabbit carotid artery:

Heparin at 1000 IU/ml was diluted 1:10 in balanced salt solution, thenused to flush and fill both cut ends of the carotid artery at the sitewhere the implant was to be grafted. Once the implant had been suturedinto place, the sutured regions were sealed with Gelfoam and then thedistal artery clamp was slowly removed allowing heparin solution toslowly enter the transplant. The proximal clamp was then slowly releasedso that heparin solution, then blood pumped from the heart, graduallyentered the transplant, pushing the heparin within the cut end throughthe transplant and in to the rest of the circulation.

After 4 months, 9 out of 10 transplants in the rabbit carotid arterywere still patent, giving a patency rate of 90% (see Table 3) as opposedto 70% for rabbit transplants in the absence of heparin.

Example 9 To Determine Whether the “Artificial Artery” is Prone orResistant to Aneurysmal Degeneration (Bursting), Intimal Hyperplasia andAtherosclerosis

The bursting strength of rabbit granulation tissue grafts, both pre- and3, 6, 9 and 12 months post-transplantation, is determined by applyingincreased intraluminal pressure via a cannula inserted at one end and amechanical pressure gauge applied to the cannulated distal end. Also,radial and uniaxial tensile loading in a FastTrack 8800J ServohydraulicTest System determines tangent Young's modulus (stiffness), yield point,ultimate strength/stress, strain to failure and tissue hysteresis. Thesevalues are compared to those of natural carotid artery. The area aroundruptures is examined histologically and the thickness of the wall inthat region, plus distal sites, measured using image analysis techniques(Mocha, Jandel Scientific). Intimal thickening, if any, at these sites(as a percent of total wall thickness) is determined and all dataanalysed by one-way ANOVA and the Turkey-Kramer multiple comparisontest.

In a separate group of rabbits (n=8), “artificial arteries” are graftedinto the right carotid artery. An excised, then sutured back into place,20 mm segment of left carotid artery acts as an internal control foreach rabbit, as manipulation will influence the degree of lipidaccumulation. The animals are fed a 1% w/v cholesterol diet for 6 weeks,then the % surface area of the implant covered with lipid-filled(Oil-Red-O staining) lesions determined by image analysis. The valuesare analysed by paired t-test. The plasma cholesterol levels of allrabbits is measured prior to the commencement of the diet and attermination.

The “artificial blood vessel” as an arteriovenous access fistula (forhaemodialysis)

The inventors graft a length of autologous “artificial blood vessel” asa femoro-femoral or brachial-cephalic arteriovenous fistula in therabbit (n=8). The effect of serial (one per week) catheterizations onthe patency and morphology of the fistula is determined at 3 months.Currently, haemodialysis patients have a similar procedure done withsaphenous vein or ePTFE, however regular catheterization leads to severedamage and graft failure. “Artificial vessels” are replaced by freshtubes of non-thrombogenic tissue grown within the patient whenevernecessary. Animals suffer no adverse effects from induction andharvesting of multiple peritoneal cavity-derived capsules over severalmonths.

Use of Tenckhoff catheter as a peritoneal dialysis catheter

Improved ways to implant and access the molding are tested using accessdevices designed for human peritoneal dialysis. Sterile silasticTenckhoff catheters (Quinton7 Instrument Co, USA) with a single ordouble cuff of polyethylene terephthalate (DACRON) to prevent migrationof bacteria and hence peritonitis, and used with or without silicondiscs to hold the omentum and bowel away from the tubing. Cut-downversions of these catheters are inserted into the rabbit peritonealcavity over a guidewire through a small incision, having first infused15 ml of 1.5% w/v dextrose dialysis solution (plus or minuscytokines/chemokines to increase the number of peritoneal macrophagespresent). The cuff is sewn in place in the peritoneum, and a Beta-Cap7adapter attached to the external portion of the catheter. This allowsperitoneal drainage or the continued addition of fluid. The catheter issurgically removed after 2 weeks, taking care not to damage thegranulation tissue capsule. This procedure allows more precise andconsistent positioning of the “artificial blood vessel” mold andalleviates invasive harvesting prior to autologous transplantation.

“Artificial blood vessel” can be genetically engineered to improveefficacy

Granulation tissue capsules are grown in the rabbit peritoneal cavity.Prior to removing the tubing and everting the tissue, the outer liningof mesothelial cells is transfected for 15 minutes in vitro with anadenoviral construct expressing tissue plasminogen activator orβ-galactosidase, with a nonviral (buffer) control group (n=8/group). Alltransfected “vessels” are grafted into the right carotid artery andtheir patency and histological appearance determined after 6 months.Transfection of vein grafts for plasminogen activator has recently beenshown to significantly reduce thrombus formation both within theengineered vein graft and downstream artery (Kuo et al, AM J. Roentgenol171:553-558, 1998).

To determine whether the “artificial blood vessel” can be grown in vitro

A vessel is created in vitro with no artificial supporting scaffold butwith a scaffold of matrix it has created itself, as with themesothelial-lined granulation tissue tube formed in the peritonealcavity of the host. An “artificial blood vessel” formed entirely invitro avoids the inconvenience to patients of having tubing insertedinto peritoneal cavity for 2 weeks.

Living cells are harvested from the rabbit peritoneal cavity through anindwelling Tenckhoff catheter. Macrophage and mesothelial cell numbersare maximised by flushing the peritoneal cavity withcytokines/chemokines and gentle agitation of the wall. Known numbers ofharvested cells are resuspended in 1:1 RPMI culture medium and rabbitperitoneal fluid then seeded onto tubes of precoated silastic or otherpolymer lining the bottom of a culture dish. The tubing is gentlyrotated after 4 hours to encourage even cell coverage. The developmentof a tube of tissue is followed histologically over the next few weeks.The everted tissue is grafted into an artery of the small animal fromwhich the cells were harvested.

A suitable catheter device is shown in FIG. 15.

Example 10 Pig Model

Before the tissue tube described herein can be grown and transplantedinto humans it is tested in a large animal model.

The pig is of a similar size to humans and has a very similarcardiovascular system, specifically the size and structure of the heartand arteries. Forty pigs (50-1000 Kg) are anaesthetised withintramuscular injection with Ketamine (15 mg/Kg)/Xylazine (1 mg/Kg) thenhalothane (1-2% v/v) in oxygen via a mask. A sterile silastic Tenckhoffcatheter (420 mm long) of outer diameter 5 mm (Quinton7 Instrument Co,USA) with a single or double cuff of polyethylene terephthalate (DACRON)to prevent migration of bacteria and hence peritonitis, is inserted intothe peritoneal cavity over a guidewire through a small incision, havingfirst infused 100 ml of 1.5% w/v dextrose dialysis solution (plus orminus cytokines/chemokines to increase the number of peritonealmacrophages present). The cuff is sewn in place in the peritoneum, and aBeta-Cap7 adapter attached to the external portion of the catheter. Thisallows the continued addition of fluid, if needed.

The catheter is surgically removed after 2 weeks taking care not todamage the granulation tissue capsule. The tissue capsule is everted andlengths of tissue tube used as bypass grafts in the anterior descendingand circumflex coronary arteries, or the carotid, iliac or femoralarteries. Patency, bursting strength, elasticity, reactivity tocontractile and relaxing agents, and histology of the grafts are testedboth pre-transplantation and 1-3 years post-transplantation.

Example 11 Eversion of Tissue Tube and Storage Prior to Transplantation

Once the tissue tube is harvested from the peritoneal cavity, it isplaced into a sterile petri dish with a scintered glass plate containingcold Hanks Balanced Salt Solution. The scintered glass plate acts toprevent the tissue tube slipping during the eversion manipulations.

There are two methods to evert the tissue tube:

1. For Lengths 40 mm and Less.

Requirements:

1× No. 4 watchmaker forceps (sterile) whose arms have been ground thin;

1× normal No. 4 watchmaker forceps (sterile).

Method:

(a) Cut both ends of the tissue capsule.

(b) Pass the arms of the ground thin watchmaker forceps through thelumen of the tube and gently grasp the distal cut end in one place.

(c) Gently pull watchmaker forceps back through the lumen, at the sametime everting the tissue with aid of the second pair of forceps.

2. For any Length of Tissue Tube.

Requirements:

Sterile tubing or filament of the same outer diameter as the tubingmold;

2× No 4 watchmaker forceps (sterile).

Method:

(a) Cut distal end of tissue capsule.

(b) Abutt a piece of sterile tubing to the uncut proximal end of tissuetube plus mold.

(c) With one pair of forceps, gently evert by pushing against cut end ofthe capsule with a second piece of silastic tubing of the same diameterat the same time threading the tissue over the second piece of tubing.Both pieces of tubing are then discarded.

The everted tissue tube can then be trimmed to the desired length andstored in cold Hanks' Balanced Salt Solution, just covering the tissueto allow maximum oxygenation, for up to 6 hours prior totransplantation.

Example 12 Preferred Method for Producing Artificial Vessels in Rabbits

The following methods and steps are employed:

1. Silastic tubing of 1.9 mm outer diameter is cut into 60 mm lengths,then spiral grooves etched into the tubing with glass paper.

2. The tubing is sterilized by soaking in 70% v/v ethanol for 4 hours,rinsed in 100% v/v ethanol then drained and air-dried in a laminar flowcabinet.

3. The tubing is then incubated overnight at 371 C in fibronectin toenhance attachment of peritoneal macrophages.

4. The animal is anaesthetised with 1.5 ml Dipravan (Propofol, 10 mg/ml,ICI Pharmaceuticals, Vic) and maintained on halothane (ICIPharmaceuticals, Vic).

5. The abdomen is shaved and surface sterilised with Chorohexidine (0.5%v/v in 70% v/v alcohol) then two pieces of the sterile tubing insertedvia a small incision into the peritoneal cavity, at the same time adding10 ml Hanks Balanced Salt Solution containing 1.5% w/v dextrose (to helpprevent adhesions) and 0.02 Fg GM-CSF (to stimulate macrophagerecruitment and proliferation).

6. The peritoneum is sutured and the animal allowed to recover.

7. After 2 weeks the animal is anaesthetised again and the tubing isharvested and placed in cold Hanks Balanced Salt Solution on a scinteredglass plate in a sterile petri dish.

8. The best of the two (or both, if multiple grafts are required)capsules of granulation tissue is everted by pushing against a cut endof the capsule with a second piece of silastic tubing of the samediameter at the same time threading the tissue over the second piece oftubing. Both pieces of tubing are then discarded.

9. The tissue is stored in shallow, cold Hanks=Balanced Salt Solutionwhile the carotid artery is exposed and a 10 mm segment removed.

10. Both of the cut ends of the carotid artery are flushed and filledwith 1000 IU/ml heparin diluted 1:10 in Hanks=Balanced Salt Solution.

11. The tissue tube is trimmed to the desired length then sutured by endto end anastomoses with 10-0 Dermalon suture (Sherwood, Davis and GeckCo, USA) between the cut ends of artery.

12. The suture points have Gelfoam (The Upjohn Co, USA) wrapped aroundthem to minimise leakage.

13. The upstream artery clamp is then gently released with some heparinflowing into the graft.

14. The downstream clamp is then gently released so that heparin andthen blood slowly enters the graft, at the same time applying gentlepressure to suture areas with Weck-cel sponge (Edward Weck Inc, USA).

15. The incision is sutured with 30 polyglycolic acid (DEXON)II (Davisand Geck, NSW), and the antibiotic Terramycin (0.3 m. of 100 mg/ml)(Pfizer, NSW) injected into the thigh muscle.

The animals nails are trimmed and taped to prevent scratching of suturesand the abdomen bandaged with Primapore.

The animal is then allowed to recover.

Example 13 Dog Model

Studies similar to those outlined herein can be performed on dogs using,for example a VASCAM device of the following specifications (see FIGS.16 and 17):

Closed ends

To prevent adhesions.

Rounded/smooth edge at distal end

To prevent injuring the bowel and/or adhesions.

Inner tube with biodegradable mesh

Polyethylene as the inner tube, most aggressive preferred (to attractcells); diameter of approximately 3.5 mm.

Polyglycolic acid (DEXON) mesh wrapped around the inner tube and held bybiodegradable suture.

Centering the inner tube

As the actual Device (length of 45-60 cm for humans; 20 cm for dogs)will have to curl to fit in the peritoneal cavity, the inner tube has tobe as centred as possible, so that there is space between inner tube andouter sheath for tissue formation.

Flange

To allow the surgeons to suture the Device to rectus sheath.

Outer sheath

Flexible, medical-grade silicone with holes that will not break withshear force.

Outer diameter of approximately 9.5 mm; and reasonable inner diameter toallow space for tissue growth.

Holes proposed to be arranged in a spiral arrangement for strength ofthe sheath.

6.4. Holes Size

Approximately 1.9558 mm.

Measurements taken manually after holes have been punched on siliconouter-sheath.

6.5. Done with a 14-Gauge Hole-Puncher.

6.6. Holes Arrangement

2 mm, 90° apart, i.e.:

Punch hole on silicon outer-sheath

Rotate silicon outer-sheath by 90°

Move puncher 2 mm in direction of sheath's length;

Punch next hole;

Repeat from step (ii).

7. Flange

7.1. No holes required on flange because surgeons could easily piercethrough flange with suture needle.

In addition to those specifications described above, the followingmodifications could also be made in relation to the VASCAM device:

Design variables to trial:

Fewer (number of) holes on silicon outer-sheath.

No holes on silicon outer-sheath near the proximal and distal ends theDevice because of high rate of adhesions through the holes, as observedfrom recent dog-experiments.

Use shorter pieces of polyglycolic acid (DEXON) mesh.

Those skilled in the art will appreciate that the invention describedherein is susceptible to variations and modifications other than thosespecifically described. It is to be understood that the inventionincludes all such variations and modifications. The invention alsoincludes all of the steps, features, compositions and compounds referredto or indicated in this specification, individually or collectively, andany and all combinations of any two or more of said steps or features.

TABLE 1 Comparison of proteins in rat granulation tissue tube, formed inthe peritoneal cavity over 2 weeks, with rat aorta using densitometricanalysis of Western blots Myofibroblast tube protein relative to aortaAntibodies (% value″ SD, n = 3) α-smooth muscle actin 108″2 β-actin 560″188 Smooth muscle myosin heavy chain  23″ 1 Vimentin  86″ 8 CollagenType I  76″ 3 Collagen Type IV  93″ 1 Elastin  9″ 1

TABLE 2 Patency of grafts without heparin with time aftertransplantation of the rat abdominal aorta or rabbit carotid artery 11.5 2 3 4 % month months months months months patent Rat (n = 30) 6/66/6 4/6 3/6 3/6 73% Rabbit (n = 20) 4/5 N/A 3/5 4/5 3/5 70%

TABLE 3 Patency of rabbit grafts WITH heparin 4 months aftertransplantation to the carotid artery 4 months % patent Rabbit (n = 10)9/10 90%

TABLE 4 Volume fraction of myofilaments of spindle shaped cells in 2week implant harvested from the peritoneal cavity of the rat and after 3months transplantation into the rat abdominal aorta Volume fraction ofmyofilaments Rat tissue (V_(v)myo) % n = 60 cells 2 week peritonealimplant 35.7″1.6  3 months post 58.7″1.4* transplantation Aorta63.7″5.7* *p < 0.1

BIBLIOGRAPHY

-   1. Campbell et al, Arteriosclerosis. 9(5): 633-43, 1989.-   2. Desmouliere et al, Journal of hematology 22: 61-64, 1995.-   3. Edwards and Roberts Clin. Mater 9: 211-223, 1992.-   4. Hartig et al, Brain-Res-Brain-Res-Protoc. 2(1): 35-43, 1997.-   5. Kleinert et al, Cell Transplant 5(4): 475-482, 1996.-   5. Koch et al, Aust. NZ. J of Surg. 67: 637-639, 1997.-   6. Kuo et al, Am. J. Roentgenol. 171: 553-558, 1998.-   7. Manderson and Campbell, Journal of Pathology 18: 77-87, 1986.-   8. Puchkov et al, Morfologia. 110(5): 15-19, 1996.-   9. Sappino et al, Lab Invest 63: 144-161, 1990.-   10. Schwartz et al, Mayo Clin Proc 68: 54-62, 1993.-   11. Verhagen et al, British Journal of Haematology 95: 542-549,    1996.-   12. Walden et al, Arch-Surg. 115(10): 1166-9, 1980.

1. A method of producing a tissue, comprising placing a molding support within a peritoneal cavity for a time and under conditions sufficient for non-vascularized tissue comprising myofibroblasts to form on the molding support and grafting the tissue into a recipient.
 2. The method of claim 1, further comprising removing the molding support from the peritoneal cavity, prior to grafting.
 3. The method of claim 1 further comprising separating the tissue from the molding support.
 4. The method of claim 3, further comprising everting the tissue.
 5. The method of claim 1, wherein the molding support is placed into the peritoneal cavity by surgical implantation.
 6. The method of claim 1, wherein the tissue is covered by non-thrombogenic mesothelial cells.
 7. The method of claim 1, wherein the molding support comprises a biodegradable matrix.
 8. The method of claim 1, wherein the molding support is a foreign body.
 9. The method of claim 8, wherein the molding support comprises silicone.
 10. The method of claim 1, wherein the molding support is a tubular molding.
 11. The method of claim 10, wherein the tissue is vascular.
 12. The method of claim 1, wherein the tissue is blood vessel.
 13. The method of claim 12, wherein the blood vessel is selected from the group consisting of an artery, an arterio-venous shunt and an access fistula.
 14. The method of claim 1, wherein the tissue is suitable for use as a substitute blood vessel.
 15. The method of claim 1, wherein the tissue is autologous relative to the recipient.
 16. The method of claim 1, wherein the recipient is human.
 17. The method of claim 1, further comprising maintaining the non-vascularized tissue in a frozen state.
 18. The method of producing a tissue, comprising placing a molding support within a peritoneal cavity for a time and under conditions sufficient for non-vascularized tissue comprising myofibroblasts to form on the molding support and grafting the tissue into a recipient, wherein the molding support is a tube and wherein the method further comprises placing the tube into the peritoneal cavity using a prosthetic device that comprises: an outer elongated tubular member having perforations or a permeable layer to permit passage of cells and fluid into and out of the tubular member; an inner elongated member removably insertable in the outer elongated member and comprising the tube around which or part of which vascular or non-vascular tissue can grow; and an external portion which is maintained on the skin surface or subcutaneously and through which the inner elongated member can be removed from the outer elongated member.
 19. The method of claim 18, wherein the outer elongated tubular membrane is comprised of silicone.
 20. The method of claim 18, wherein the perforations are about 2 mm in diameter. 